Toxoplasma gondii is a major food borne pathogen in the US, a leading cause of infectious retinitis in children and can be lethal to the fetus and immuno-compromised adults. To date there is no vaccine, and while there are a limited number of existing therapeutics they have undesirable side-effects and do not cure the infection. Therefore the development of new therapies would reduce human suffering associated with this pathogen. In the post-genomic era, the challenge is to identify those gene products that may be successfully targeted for therapeutic intervention. Driven by recent innovations in the analysis of protein complexes through a state-of- the-art co-fractionation proteomics platform developed by the applicants (Havugimana et al., Cell 2012; Wan et al., Nature 2015; 2,3), this project will systematically identify native protein complexes for two key life cycle stages from two strains of T. gondii (AIM 1). Network-based comparisons will reveal how proteins are orchestrated to form complexes required not only for invasion but also for modulating host pathways, processes key for parasite transmission and persistence. Further, comparisons between strains will identify both core- conserved elements, indicating common functionality, as well as lineage-specific innovations with the potential to impact parasite virulence. To validate our findings, we will perform detailed functional characterization of proteins with newly predicted roles in host modulation pathways (AIM 2). From our preliminary data, we have already selected two promising candidates to begin validation. These will be supplemented with additional proteins with predicted effector roles identified through our network analyses. Together this program of research promises to deliver unique biological insights into the organization of the complexes and pathways that drive parasite invasion and persistence, revealing novel parasite virulence mechanisms that will drive new strategies for designing potent anti-parasitics.